Breakthrough candidemia with hematological disease: Results from a single-center retrospective study in Japan, 2009–2020

Abstract Breakthrough candidemia (BrC) is a significant problem in immunocompromised patients, particularly those with hematological disorders. To assess the characteristics of BrC in patients with hematologic disease treated with novel antifungal agents, we collected clinical and microbiological information on said patients from 2009 to 2020 in our institution. Forty cases were identified, of which 29 (72.5%) received hematopoietic stem cell transplant (HSCT)-related therapy. At BrC onset, the most administered class of antifungal agents were echinocandins, administered to 70% of patients. Candida guilliermondii complex was the most frequently isolated species (32.5%), followed by C. parapsilosis (30%). These two isolates were echinocandin-susceptible in vitro but had naturally occurring FKS gene polymorphisms that reduced echinocandin susceptibility. Frequent isolation of these echinocandin-reduced-susceptible strains in BrC may be associated with the widespread use of echinocandins. In this study, the 30-day crude mortality rate in the group receiving HSCT-related therapy was significantly higher than in the group not receiving it (55.2% versus 18.2%, P = .0297). Most patients affected by C. guilliermondii complex BrC (92.3%) received HSCT-related therapy and had a 30-day mortality rate of 53.8%; despite treatment administration, 3 of 13 patients had persistent candidemia. Based on our results, C. guilliermondii complex BrC is a potentially fatal condition in patients receiving HSCT-related therapy with echinocandin administration.


Introduction
Invasive fungal infections (IFIs) are life-threatening issues in immunocompromised hosts, particularly patients with hematological disease. Thus, antifungal prophylaxis is regularly used in patients with hematological disease to prevent IFIs. [1][2][3][4] These antifungal treatments can decrease the prevalence of IFI and IFI-related mortality. [5][6][7][8][9] However, since the 1990s, they have resulted in breakthrough IFIs, 10 , 11 which need more attention because new antifungal therapies, such as prophylaxis and novel immunomodulating agents, make diagnosing IFIs difficult. [12][13][14] Breakthrough candidemia (BrC) is one of the most important and prevalent breakthrough IFIs. Despite numerous reports on the epidemiology, risk factors, and prognosis of BrC, [15][16][17][18][19][20][21][22] the epidemiology of IFIs has changed since newer antifungal agents have become available, 23 particularly those of the echinocandin class. 24 In addition, emergence of some resistant Candida species, such as C . glabrata and C . auris , 25 , 26 and regional differences in resistance rates [27][28][29] may influence the epidemiology of BrC. Indeed, several cases of BrC caused by the rare pathogen C. guilliermondii complex have been identified in hematological patients at our institution in recent years. Candida guilliermondii complex is an uncommon Candida species rarely observed to cause fungemia. 30 Apart from case reports and small surveys, 15 , 20 , 31-33 there is little information regarding BrC involving C. guilliermondii complex among patients with hematological disease. Therefore, to clarify the clinical and microbiological information of BrC, particularly associated with respect to the C. guilliermondii complex, we conducted a retrospective study on BrC in hematological patients at our institution from 2009 to 2020.

Patients and data collection
We conducted a retrospective analysis on BrC among patients with hematological disease admitted at Kyushu University Hospital (a 1275-bed tertiary teaching hospital in Fukuoka, Japan) between January 2009 and December 2020. We included inpatients undergoing treatments for hematological disease and related conditions, including graft-versus-host disease. Medical records were reviewed for collection of the following clinical information: underlying hematological disease and treatment, clinical symptoms at BrC onset, prior antifungal exposure, antifungal treatment, and outcomes after BrC diagnosis, risk factors for candidemia, including use of corticosteroid and other immunosuppressive agents, central venous catheter (CVC) insertion, abdominal operation, intensive care unit (ICU) stay, use of broad-spectrum antimicrobial agents, and neutropenia. This study was approved by the institutional review board of Kyushu University Hospital (approval no. 22223-00).

Definitions
Candidemia was defined as the isolation of any Candida species from ≥1 blood culture set from a patient with signs and symptoms of infection. BrC was defined as candidemia in patients receiving systemic antifungal agents for ≥3 days before the first positive blood culture. 34 If the same Candida species was detected within 4 weeks in the same patient, then it was considered to be the same episode. 35 , 36 Sustained candidemia was defined as candidemia persisting for > 2 weeks. Recurrence of candidemia was considered when a blood culture became negative and the same Candida species was detected again > 4 weeks after the initial detection. Septic shock was defined as sepsis-induced hypotension persisting despite adequate fluid resuscitation. 37 The serum (1, 3)-beta-D-glucan (BDG) level at onset was defined as the BDG level measured within 3 days before and after the onset of candidemia. The Wako turbidimetric assay (Wako Pure Chemical Industries, Tokyo, Japan) was used to measure the serum BDG level using the cutoff values for positivity recommended by the manufacturer (11 pg/ml). 15 Neutropenia and severe neutropenia were defined as absolute neutrophil counts of ≤500 cells/ μl and ≤100 cells/ μl, respectively. 15 Broad-spectrum antimicrobial agents included carbapenem, fourth-generation cephalosporin, and piperacillin/tazobactam. The choice of antifungal treatment was under the discretion of each physician in all cases.

Microbiological tests
Blood culture samples were processed using a BacT/Alert system (bioMérieux Japan Ltd, Tokyo, Japan, from January 2009 to March 2013) and a BACTEC FX system (Nippon Becton Dickinson Company Ltd, Tokyo, Japan, from April 2013 to December 2020). Positive samples for yeast according to a gram stain were subcultured on Sabouraud dextrose agar (Nippon Becton Dickinson Company, Ltd, Japan) at 35 • C for 48 h. These isolates were initially identified by VITEK 2 YST ID Card (bioMérieux Japan Ltd, from January 2009 to March 2013) and matrix-assisted laser desorption/ionization timeof-flight MALDI VITEK MS (bioMérieux Japan Ltd, from April 2013 to December 2020). Breakthrough Candida isolates were stored at −80 • C, and molecular identification was conducted by sequencing of the internal transcribed spacer region according to the CLSI MM18-A protocol.
An antifungal susceptibility test was performed using broth microdilution methods with a commercial kit ASTY (Kyokuto Pharmaceutical Industrial, Tokyo, Japan) following the manufacturer's instructions. For the four species ( C . albicans , C . glabrata , C. krusei , and C. parapsilosis ), minimal inhibitory concentration (MIC) results were interpreted following the Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) according to the CLSI M27-S4 standard. For agents without species-specific CBPs, epidemiological cutoff values (ECVs) were applied. 38 The MIC interpretation for C. nivariensis was determined according to CLSI standards for C. glabrata . In this study, we defined non-susceptible strains as those whose susceptibility test results were determined other than susceptible by CBPs and non-wild type by ECVs. Candida parapsilosis ATCC 22019 was tested as a quality control for every new lot.
In addition, we performed screening of mutations in hot spot regions of FKS genes. The FKS1 hot spot 1 (HS1) and hot spot 2 (HS2) regions were sequenced for all breakthrough Candida isolates. In addition, FKS2 HS1 and HS2 regions were also analyzed in C. glabrata . Polymerase chain reaction amplification was conducted using two sets of primers previously described ( https:// doi.org/ 10.3030/ 642095 ). 25 , 39 Statistical analysis Continuous data are expressed as the median and interquartile range (IQR), and categorical data are described by the count and percentage. The Mann-Whitney U -test was used to compare continuous variables, and the Fisher's exact test was used to compare categorical variables. The survival curves at 30 days were calculated using the Kaplan-Meier method, and differences between curves were evaluated using the log-rank test. A P -value < .05 was considered statistically significant. All statistical tests were conducted using EZR. 40

Characteristics of BrC in hematological patients
From January 2009 to December 2020, 48 episodes of fungemia (44 candidemia, 2 fusariosis, and 2 trichsporonemia) were detected in patients with hematological disease at Kyushu University Hospital. A total of 40 cases of candidemia in 39 patients met the definition of BrC. Only one patient developed two episodes of BrC (due to C. lusitaniae and C. parapsilosis , respectively) during the study period. The clinical characteristics of the 40 episodes are shown in Table 1

Antifungal susceptibility to previously administered agents
Six of eight BrC cases (75%) under azole therapy were caused by Candida species not susceptible to the preceding azoles in vitro ( Table 4 ). The remaining two patients received low-dose FLC (100 or 200 mg/day, Table 4 ); in both cases, FLC-sensitive C. albicans was isolated. On the other hand, most BrCs during echinocandin treatment (23/28, 82.1%) were caused by Candida species susceptible to the echinocandins used; all eight BrCs during L-AMB administration were due to AMBsensitive Candida species. The trend in susceptibility of BrC isolates to the prior drug was that azole-resistant strains were more common in the azole pre-treated group, whereas isolates from echinocandin pre-treated cases and L-AMB pre-treated cases were often sensitive to the prior drug, consistent with previous reports. 15-17 , 19 , 20 , 32 , 41 The susceptibility to prior echinocandin treatment depends on Candida species. All isolates of the C. guilliermondii complex (9 isolates), C. parapsilosis (12 isolates), C. krusei (1 isolate), and C. lusitaniae (1 isolate) were susceptible to echinocandins, whereas C. nivariensis (2 isolates), C. albicans (1 isolate), C. glabrata (1 isolate), and C. kefyr (1 isolate) were not. Since reduced susceptibility to echinocandin involves amino acid substitutions in the hot spot region of the FKS protein, the catalytic subunit of the 1,3-β-D-glucan synthase, 25 , 42-45 we evaluated the presence of FKS gene mutations in isolates. Each strain of C . glabrata , C . albicans , and C. kefyr isolated during echinocandin therapy showed a mutation in the HS1 region of FKS1 and non-susceptibility to prior echinocandins ( Table 4 ). All C. guilliermondii complex and    '… ' = not tested. C. parapsilosis isolates harbored naturally occurring polymorphisms in the FKS1 region, showing a tendency toward higher echinocandin MICs than wild-type C. albicans , as previously reported. [42][43][44][45] Treatments and outcomes after BrC diagnosis A detailed description on treatments and outcomes is shown in Table 4 . A total of 38 cases (95%) were diagnosed with BrC; two (5%) patients died before the diagnosis of breakthrough infection. The crude mortality rates at days 14 and 30 were 32.5% and 45.0%, respectively. The median time from BrC to death within 30 days was 9.5 days (IQR, 3-15 days). Thirtythree cases (86.8%) changed the antifungal therapy after BrC, while five (13.2%) continued with the prior antifungal agents. One case treated with VRC died before BrC diagnosis; the isolates were later confirmed to be resistant to VRC. The prior azole treatments of the other five cases were changed to therapies comprising susceptible antifungal agents; however, three (60%) patients died within 30 days after BrC. In addition, 21 (87.5%) BrC patients with prior echinocandin monotherapies received L-AMB-based regimens, including combination therapies; of those, 1/3 died within 30 days of diagnosis. Two patients (8.3%) continued with their previous MCF treatment because of the good susceptibility and clinical course. The last case (4.2%) was continuously treated with CPF and received FLC after diagnosis, resulting in a favorable outcome. Five of six prior L-AMB treatment cases (83.3%) were added or changed to echinocandin agents with or without VRC after diagnosis; two of whom died. One case receiving L-AMB treatment died before BrC diagnosis, although the isolate was sensitive to amphotericin B. One BrC case receiving combination therapy was treated with L-AMB monotherapy after the prior treatment proved ineffective, with a fatal consequence. Two BrC cases in which one of the two prior antifungal drugs was ineffective were either switched to another class of agents or continued on the prior medication, but both died. The remaining BrC case on combination therapy changed treatment from L-AMB to VCZ on a decision of ineffective previous therapy and survived, although both previous medications were susceptible to the isolate. The patients that developed BrC during combination therapy had a worse prognosis, with 3 out of 4 patients dying, possibly because they were more severely ill, given that all patients received HSCT-related therapy.

Features of C. guilliermondii and C. parapsilosis breakthrough infection
Since the C. guilliermondii complex and C. parapsilosis were the two dominant species in this study (Table 2 ), we focused on the characteristics of breakthrough infection caused by these two species (  Table 4 ). Moreover, the No. 35 case relapsed after one month. The incidence of septic shock at BrC onset was similar in both C. guilliermondii complex and C. parapsilosis groups (15.4% and 16.7%, respectively), as was the presence of disseminated infections (7.6% and 8.3%, respectively). The positive rate of serum BDG was 50% in the C. parapsilosis cohort, same as for all BrC cases in this study (Table 1 ), while that in the C. guilliermondii complex cohort was lower (30.8%). Echinocandin monotherapy was the most frequent prior treatment of both C. parapsilosis and C. guilliermondii complex BrC; in particular, all 12 C. parapsilosis BrC episodes occurred while using echinocandins. On the other hand, the C. guilliermondii complex was isolated in 13 patients treated with all classes of antifungal agents (MCF, 5; CPF, 2; L-AMB, 3; VCZ, 1; CPF plus VCZ, 1; and CPF plus L-AMB, 1; respectively). Median time of prior antifungal agent use was similar between groups ( C. guilliermondii complex, 30 days; C. parapsilosis , 27 days, respectively).
The survival curves at 30 days of both groups, calculated using the Kaplan-Meier method (Fig. 1 a), show that the 30day survival rate of the C. parapsilosis group was higher-but not significantly different-than that of the C. guilliermondii complex (83.3%, 46.2%, respectively, P = .054).

Mortality in BrC patients with and without HSCT-related therapy
The high mortality of C. guilliermondii BrC (53.8%) was associated with a higher rate of HSCT treatment (92.3%) than in the C. parapsilosis cohort (Fig. 1 a and Table 5 ). Furthermore, all BrC cases observed during combination therapy also received HSCT-related treatment. Given the high mortality rate (75%), to determine whether HSCT was relevant to the BrC outcomes, we evaluated the impact of HSCTrelated therapy on BrC survival. BrC patients receiving HSCTrelated treatment had a significantly lower survival at 30 days than the non-HSCT cohort (Fig. 1 b, 44.8% versus 81.8%, P = .0297).

Discussion
In this study, we described the clinical and microbiological features of 40 BrC cases in patients with hematologic diseases at a single tertiary institution. The most frequent isolate was the C. guilliermondii complex (32.5%), and the second was C.  parapsilosis (30.0%). To the best of our knowledge, the unfavorable characteristics of the C. guilliermondii complex BrC in hematological patients are highlighted for the first time in this study. Candida guilliermondii complex is an uncommon yeast, responsible for only 1.4% of all candidemia cases. 30 This proportion depends on the geographic region, with relatively higher rates in Asia (1.8%-2.6%) and Latin America (3.7%). 30 , 31 , 46 The proportion of C. guilliermondii complex is slightly higher when it is the causative agent of BrC (4.8%-15%). 15,16,20,34 Most C. guilliermondii complex candidemia has been reported in patients with cancer, especially those with hematological malignancies. 15 , 16 , 31 , 34 , 47-49 This might explain why C. guilliermondii complex was found in most patients in our study. However, our data showed a higher incidence than previously reported (4.8%-15%), 15 , 16 , 20 , 34 which may be due to regional characteristics or other factors. The Japanese national surveillance data on candidemia (2010-2019) show an increasing frequency of infection with C. guilliermondii since 2014. 49 In addition, the incidence rate of candidemia caused by C. guilliermondii complex and C. parapsilosis in Japanese hospitals providing HSCT was significantly higher than that in other hospitals. 49 In our study, C. guilliermondii complex BrC with hematological disease was detected in 2011 (Fig. S1). Although there was no clear upward trend in C. guilliermondii complex BrC, the combined proportion of C. guilliermondii complex and C. parapsilosis BrC showed an increasing trend from 2011 onwards. The proportion of C. guilliermondii complex in BrC with hematological disease in this study was higher than that of C. guilliermondii complex in all candidemia in our hospital over the same period, 2009-2020. Thus, our single-center data are consistent with the general trend in Japan.
In our study, breakthrough infections with C. guilliermondii complex tended to be more common in HSCT patients (92.3%) and showed a high mortality rate (53.8%); there were three cases (23.1%) of C. guilliermondii complex candidemia lasting > 2 weeks, of which one case relapsed after one month, suggesting that these refractory fungemia cases might be related to severe host conditions, especially HSCT, and that the breakthrough infection may not be the direct cause of death. Indeed, the mortality rate for BrC in HSCT recipients was significantly higher than in the non-HSCT cohort ( P = .0297, Fig. 1 b). Candida guilliermondii complex BrC in hematological patients may be a potentially fatal condition, including critical underlying disease. A previous report 50 showed that C. guilliermondii complex candidemia was more frequently persistent than that of C. albicans ; moreover, patients with C. guilliermondii complex candidemia had usually a severe underlying disease but a lower mortality rate and less severe clinical presentations than those with C. albicans . This is mostly consistent with our results, except for the lower mortality rate; the reason for the higher mortality rate in our patients could be that 92.3% were receiving HSCT-related treatment and may have had more severe underlying conditions. Previously, C. albicans was the most frequently isolated species, but the widespread FLC use has led to increased isolation rate of relatively azole-resistant Candida species, such as C. glabrata and C. krusei . 11 , 17 , 51-56 Over the last decade, C. parapsilosis has been reported as the most common causative organism (41%-56%) in BrC observed during echinocandin administration, especially in hematological patients. 15 , 57 , 58 In this study, echinocandins were the most common antifungal agents administered at BrC onset; 60% of all patients received echinocandin monotherapy and 10% combination therapy including echinocandins (Tables 2 and 4 ). Furthermore, C. parapsilosis was the second most common pathogen causing BrC (30%) ( Table 3 ). The high frequency of BrC caused by the C. guilliermondii complex and C. parapsilosis in our study could be attributed to their intrinsically low echinocandin susceptibility and the high frequency of echinocandin use. In this study, all cases of BrC due to C. parapsilosis developed during echinocandin administration as did 9 of 13 cases of BrC due to the C. guilliermondii complex; the remaining four cases with the C. guilliermondii complex received echinocandins as prior therapy within the previous 60 days. All C. guilliermondii complex and C. parapsilosis isolates harbored naturally occurring polymorphisms in the FKS1 region and showed a tendency toward higher echinocandin MICs than wild-type C. albicans , as previously reported. [42][43][44][45] All these isolates were echinocandin-sensitive according to CLSI M60-Ed2 criteria. However, since they caused BrC, there might be no relation between the efficacy in vivo and MIC in vitro , particularly in hematological patients. All C. guilliermondii complex isolates in this study had the naturally occurring L633M and T634A substitutions as intrinsic hot spot mutations in FKS1 , consistent with previous reports. 44 The positive BDG rate was low in the C. guilliermondii complex BrC, 4 out of 13 cases (30.8%); thus, these FKS1 intrinsic mutations and the use of antifungal drugs such as echinocandin could potentially affect BDG synthesis. 59 Future research is needed to clarify this point.
This study had some limitations. It was retrospectively conducted at a single tertiary-care teaching hospital. Thus, its results may not be generalizable to other institutions. In addition, the number of cases was relatively small. More cases need to be accumulated to evaluate prognosis according to Candida species and to analyze the clinical characteristics of BrC patients who received HSCT-related therapy.